The present invention relates to novel banana fruit-associated promoters, a melon actin promoter and a banana fruit-associated/melon actin fusion promoter. The invention also relates to heterologous nucleic acid constructs, vectors, kits, and transformation methods employing such promoters. The invention further relates to transgenic plant cells and plants transformed with heterologous nucleic acid constructs comprising the promoters and methods for screening plant promoters in various types of plant tissue using a transient expression assay.
Adams, D. O., and Yang, S. F., Plant Physiology 70:117-123 (1977).
Altschul, et al., Nucl. Acids Res. 25(17) 3389-3402 (1997).
An, G, et al., EMBO J. 4:277-284 (1985).
An, Y Q et al., Plant J. 10(1):107-21 (1996).
Ausubel, F M, et al., in Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Media, Pa. (1992).
Ayub, R., et al., Nature Biotechnology 14:862-866 (1996).
Becker, D., et al., Plant Mol. Biol. 20:1195-1197 (1992).
Bellini, C., et al., Bio/Technology 7(5):503-508 (1989).
Bestwick, R K, et al., PCT International Publication No. WO 95/35387, published Dec. 28, 1995.
Brunke, K J and Wilson, S L, European Patent Publication No. 0 559 603 A2, published Sep. 08, 1993.
Clendennen, S K and May, Plant Physiol. 115:463-469 (1997).
Comai, L. and Coning, A. J., U.S. Pat. No. 5,187,267, issued Feb. 16, 1993.
Cordes, S, et al., The Plant Cell 1:1025-1034 (1989).
Dominguez-Puigjaner et al., Plant Physiol. 114:1071-1076 (1997).
Dong, J. Z., et al., Bio/Technology 9:858-863, 1991.
Fang, G, and Grumet, R, Plant Cell Rep. 9:160-164 (1990).
Ferro, A, et al., U.S. Pat. No. 5,416,250, issued May 16, 1995.
Frisch et al., Plant Mol. Biol. 27:405-409, 1995.
Gonsalves, C, et al., J. Amer. Soc. Hort. Sci. 119:345-355 (1994).
Hooykaas, P J, and Schilperoot, R A, in Trends in Biochemical Sciences, International Union of Biochemistry and Elsevier Science Publishers, v.10(8):307-309 (1985).
Houck, C M and Pear, J R, U.S. Pat. No. 4,943,674, issued Jul. 24, 1990.
Hughes, J A, et al., J. Bact. 169:3625-3632 (1987).
Jefferson, R A, et al., EMBO J. 6:3901 (1987a).
Jefferson, R A, Plant Mol. Biol. Rep. 5:387 (1987b).
Jefferson, R A, Nature 342(6251) 837-838, 1989).
Klein, T. M., et al., PNAS(USA) 85(22):8502-8505 (1988).
Leisner, S M, and Gelvin, S B, Proc. Natl. Acad. Sci. USA 85(8):2553-2557 (1988).
Lin, E et al., Plant Mol. Biol. 23:489-499 (1993).
Maniatis, T et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
McCormick et al., Plant Cell Reports 5:81-84, 1986.
McElroy D, et al., Plant Mol Biol 15(2):257-68 (1990).
Medina-Escobar et al., Plant Mol Biol 34:867-877 (1997).
Medina-Suarez et al., Plant Physiol 115:453-461 (1997).
Miki, B. L. A., et al., Plant DNA Infectious Agents (Hohn, T., et al., eds.) Springer-Verlag, Wien, Austria, pp.249-265 (1987).
Ni, M et al., Plant J. 7:661-676 (1995).
Norelli et al., HortScience, 31:1026-1027, 1996.
Pearson and Meagher, Plant Mol Biol 14(4):513-26, 1990.
Picton, S, et al., Plant Physiology 103(4):1471-1472, 1993.
Ranier et al., Bio/Technology 8:33-38, 1990.
Robinson, H L and Torres, Calif., Sem. Immunol. 9:271-282, 1997.
Rogers, S, U.S. Pat. No. 5,034,322, issued Jul. 23, 1991.
Sagi et al., Bio/Technology 5:481-485, 1995.
Sambrook, J, et al., in MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Vol. 2 (1989).
Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980).
Tommerup, H, et al., Eur. Congr. Biotechnol. 5:916-918 (1990).
Valles, M P and Lasa, J M, Plant Cell Rep. 13:145-148 (1994).
Van Haaren, M J J, et al., Plant Mol. Bio. 21:625-640 (1993).
Verdaguer et al., Plant Mol Biol. 37:1055-1067, 1998
Wang et al., Mol Cell Biol. 12(8):3399-406 (1992).
Yoshioka, K, et al., Jpn. J. Breeding 42(2):278-285 (1992).
Zhu, Q, et al., Plant Cell 7:1681-1689 (1995).
Transcriptional regulatory sequences or promoters that regulate gene expression in plants are essential elements of plant genetic engineering. Several examples of promoters useful for the expression of heterologous genes in plants are now available (Zhu, et al., 1995; Ni, et al., 1995).
Most promoters are from about 500-1500 bases. Promoters for expressing a heterologous gene sequence in plants can be derived from plant DNA, e.g., the cauliflower heat shock protein 80 (hsp80, Brunke and Wilson, 1993; U.S. Pat. No. 5,612,472), or from other sources, for example, plant viruses e.g., the 35S cauliflower mosaic virus promoter, or bacteria which infect plants, e.g., the nopaline synthase (nos) promoter (Rogers, 1991), the octopine synthase (ocs) promoter (Leisner and Gelvin, 1988) and the mannopine synthase (mas) promoter from Agrobacterium.
Expression of heterologous genes or selected sequences of genes in transgenic plants has typically involved the use of constitutive promoters, which drive the expression of a product throughout the plant at all times and in most tissues (e.g., hsp80), the tomato ubiquitin promoter (Picton, et al., 1993), and the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393; and 5,783,394).
A limited number of inducible and/or tissue specific promoters are known. Promoters that provide fruit-specific expression include the E4 and E8 promoter from tomato (Cordes, et al., 1989; Bestwick, et al., 1995; U.S. Pat. No. 5, 859,330). Another fruit-specific promoter is the tomato 2A11 gene promoter. It has been demonstrated that nucleic acid sequences placed under the regulatory control of the 5xe2x80x2 non-coding region of the tomato 2A11 gene (Van Haaren, 1993) are preferentially transcribed in developing fruit tissue. Fruit specific regulation of the kiwifruit actinidin promoter has been reported to be conserved in transgenic petunia plants (Lin, et al., 1993).
Pectate lyase (PEL) has been previously identified as fruit- and ripening-associated in banana (Dominguez-Puigjaner et al., 1997; Medina-Suarez et al., 1997), and has recently been associated with breakdown of cell wall components and subsequent fruit softening during strawberry fruit ripening (Medina-Escobar et al., 1997).
Ethylene is a plant hormone influencing many aspects of plant growth and development, and is known to play a major role in the ripening process in fruits and vegetables. A large amount of ethylene is also produced following trauma caused by chemicals, temperature extremes, water stress, ultraviolet light, insect damage, disease, or mechanical wounding. In some tissues, exposure to only a small amount of ethylene may cause an avalanche of ethylene production in adjacent plants or plant tissues such as fresh produce. This autocatalytic effect can be very pronounced and lead to loss of fruit quality during transportation and storage.
In plants, methionine is converted to AdoMet, which is converted to ACC, which is converted to ethylene. AdoMet is synthesized via a condensation reaction between methionine and Adenosine triphosphate (ATP). A bacterial enzyme, AdoMet hydrolase (AdoMetase), which is normally not present in plant tissue, hydrolyzes AdoMet to homoserine and MTA, both of which are recycled to methionine. Plant transformation vectors, tomato fruit-specific promoters and methods of transforming plants with heterologous nucleic acid constructs effective to express AdoMetase (also termed xe2x80x9cSAMasexe2x80x9d) in plant cells and thereby modulate ethylene expression, have been described. See, e.g. co-owned U.S. Pat. Nos. 5,416,250; 5,589,623; 5,723,746; 5,750,864; and 5,859,330, expressly incorporated by reference, herein.
A need exists for constitutive promoters of plant origin and for plant promoters that are functional in fruit, and are capable of providing high level expression of heterologous genes in the cells of fruit.
Applicants have identified novel banana fruit-associated promoters designated in the present application as xe2x80x9cTRXxe2x80x9d and xe2x80x9cPELxe2x80x9d, a melon actin promoter, designated xe2x80x9cmACTINxe2x80x9d and melon actin:TRX fusion promoters designated xe2x80x9cTRX-intronxe2x80x9d and xe2x80x9cTRX-actinxe2x80x9d.
In one embodiment, the invention provides an isolated nucleic acid molecule comprising a banana fruit-associated TRX or xe2x80x9cG1Axe2x80x9d promoter. In one aspect of this embodiment, the isolated nucleic acid comprises nucleotides 13 to 990 of SEQ ID NO:1 (presented as SEQ ID NO:2), or a functional portion thereof, or is complementary to the nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
In another embodiment, the invention provides an isolated nucleic acid molecule comprising a banana fruit-associated PEL promoter. In one aspect of this embodiment, the isolated nucleic acid comprises the sequence presented as SEQ ID NO:3 (FIGS. 3A and B), or a functional portion thereof (e.g., nucleotides 564-2010 or 1099-2010 of SEQ ID NO:3), or is complementary to the nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions
In another embodiment, the invention provides an isolated nucleic acid molecule comprising a melon actin promoter designated xe2x80x9cmACTINxe2x80x9d. In one aspect of this embodiment, the isolated nucleic acid comprises the cDNA sequence presented as SEQ ID NO:4 (FIG. 4), or is complementary to the nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
In a related embodiment, the invention provides a TRX-monocot intron and a TRX-actin fusion promoter, designated xe2x80x9cTRX-intronxe2x80x9d and xe2x80x9cTRX-actin, respectively. In one aspect, the isolated nucleic acid comprises the TRX-intron fusion promoter sequence presented as SEQ ID NO:5 (FIGS. 5A-B), or is complementary to the nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions. In another aspect, the isolated nucleic acid comprises the TRX-actin fusion promoter sequence presented as SEQ ID NO:6 (FIG. 6), or is complementary to the nucleic acid sequence, and remains stably bound to it under at least moderate, and optionally, under high stringency conditions.
The invention also provides nucleic acid constructs having a DNA coding sequence under the transcriptional control of a banana fruit-associated promoter, a melon actin promoter, a TRX intron fusion promoter or a banana TRX melon actin fusion promoter. The DNA coding sequence is typically heterologous to the promoter and is operably linked to the promoter to enable expression of the encoded product in plant cells.
In one respect, the banana fruit-associated, TRX-intron and TRX-actin promoters of the present invention can be used to express heterologous genes in a fruit-specific manner. In a related aspect of the invention, such promoters may be used to modulate ethylene production in transformed fruit cells and to thereby alter the ripening phenotype of transgenic fruit comprising such fruit cells.
In another respect, the melon actin promoter of the present invention can be used to express heterologous genes in transformed plant cells, of either dicot or monocot origin.
In a related aspect, the melon actin promoter can be used to consitituitively express heterologous genes in tissue of dicot or monocot plants.
The invention further includes a method for producing a transgenic plant such as a fruit-bearing plant. In this method, the chimeric gene of the present invention, typically carried in an expression vector allowing selection in plant cells, is introduced into progenitor cells of selected plant. These progenitor cells are then grown to produce a transgenic plant bearing fruit.
In a further related embodiment, the invention includes a plant cell, plant tissue, transgenic plant, fruit cell, whole fruit, seeds or calli containing any of the above-described promoters, as well as plant cells comprising the promoters and/or gene products expressed under the control of the promoters.
In another embodiment, the invention provides a transient expression method for evaluating promoter expression in plant tissue. In one preferred aspect of this embodiment, a nucleic acid construct comprising a candidate promoter sequence operably linked to a GUS reporter gene is assembled, plant tissue is prepared for transformation, the nucleic acid construct is introduced into the prepared plant tissue and the plant tissue is cultured under conditions effective and for a time sufficient to detect expression of the transgene.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples.